Mechanisms Regulating Mitochondrial Transfer in Human Corneal Epithelial Cells

P URPOSE . The intraepithelial corneal nerves (ICNs) innervating the cornea are essential to corneal epithelial cell homeostasis. Rho-associated kinase (ROCK) inhibitors (RIs) have been reported to play roles in neuron survival after injury and in mitochondrial transfer between corneal epithelial cells. In this study, the mechanisms human corneal limbal epithelial (HCLE) cells use to control intercellular mitochondrial transfer are assessed. M ETHODS . Mitotracker and AAV1 mitotag eGFPmCherry were used to allow us to study mitochondrial transfer between HCLE cells and neurons in co-cultures and in HCLE cultures. A mitochondrial transfer assay was developed using HCLE cells to quantify the impact of cell stress and inhibition of phagocytosis, gap junctions, and ROCK on mitochondrial transfer, cell adhesion, migration, matrix deposition, and mitochondrial content. R ESULTS . Bidirectional mitochondrial transfer occurs between HCLE cells and neurons. Mitochondrial transfer among HCLE cells is inhibited when gap junction function is reduced and enhanced by acid stress and by inhibition of either phagocytosis or ROCK. Media conditioned by RI-treated cells stimulates cell adhesion and mitochondrial transfer. C ONCLUSIONS . Maximal mitochondrial transfer takes place when gap junctions are functional, when ROCK and phagocytosis are inhibited, and when cells are stressed by low pH media. Treatments that reduce mitochondrial content increase HCLE cell mitochondrial transfer. ROCK inhibition in co-cultures causes the release and adhesion of mitochondria to substrates where they can be engulfed by migrating HCLE cells and growing axons and their growth cones.